The invention relates to anti-freeze proteins (AFPs) and frozen food product containing AFPs.
Anti-freeze proteins (AFPs) have been suggested for improving the freezing tolerance of foodstuffs.
For the purpose of the invention, the term AFP has the meaning as well-known in the art, namely those proteins which exhibit the activity of inhibit the growth of ice crystals. See for example U.S. Pat. No. 5,118,792.
WO 90/13571 discloses antifreeze peptides produced chemically or by recombinant DNA techniques. The AFPs can suitably be used in food-products.
WO 92/22581 discloses AFPs from plants which can be used for controlling ice crystal shape in ice-cream. This document also describes a process for extracting a polypeptide composition from extracellular spaces of plants by infiltrating leaves with an extraction medium without rupturing the plants.
WO 94/03617 discloses the production of AFPs from yeast and their possible use in ice-cream. WO 96/11586 describes fish AFPs produced by microbes.
Several literature places also mention the isolation and/or use of plant proteins for cryoprotection. Cryoprotective proteins have a function in the protection of plant membranes against frost damage. These proteins, however, do not possess recrystallisation inhibition properties and are, therefore, not embraced within the terms AFPs.
Hincha in Journal of Plant Physiology, 1992, 140, 236-240 describes the isolation of cryoprotective proteins from cabbage. Volger in Biochimica et Biophysica Acta, 412 (1975), 335-349 describes the isolation of cryoprotective leaf proteins from spinach. Boothe in Plant Physiol (1995), 108: 759-803 describes the isolation of proteins from Brassica napus. Again, these proteins are believed to be cryoprotective proteins rather than AFPs. Neven in Plant Molecular Biology 21: 291-305, 1993 describes the DNA characterisation of a spinach cryoprotective protein. Salzman in Abstracts and Reviews of the 18th Annual Meeting of the ASEV/Eastern Section in Am. J. Enol. Vitic., Vol. 44, No. 4, 1993 describes the presence of boiling-stable polypeptides in buds of Vitis. Although the proteins are analogous to fish antifreeze peptides, they are cryoprotective proteins and not AFPs. Lin in Biochemical and Biophysical Research Communication, Vol. 183, No. 3, 1992, pages 1103-1108 and in Lin, Plant Physiology (1992) 99, 519-525 describes the 15 kDa cryoprotective polypeptide from Arabidopsis Hakaira. Houde in The Plant Journal (1995) 8(4), 583-593 mentions cryoprotective proteins from wheat.
Up till now, however the use of AFPs has not been applied to commercially available food products. One reason for this are the high costs and complicated process for obtaining AFPs. Another reason is that the AFPs which until now have been suggested for use in frozen food products cannot be incorporated in the standard formulation mix, because they tend to destabilise during processing especially during the pasteurisation step. This destabilisation is believed to be caused by the denaturation of the AFPs; this is a well-known effect commonly observed for peptides and proteins.
In our non pre-published patent application: WO 98/4148 it has been described that particularly good AFPs can be isolated from natural sources such as Lichen.
Applicants have now been able to determine the partial amino acid sequence of a particularly active AFP from Lichen.
Accordingly the invention relates to an AFP which can be derived from Lichen, said AFP having an apparent molecular weight of about 24 kDa and an amino acid sequence from the N-terminus of: A-P-A-W-M-D-A-E-S-F-G-A-I-A-H-G-G-L (SEQ ID NO:1).
Also embraced in the scope of our invention are proteins having a sequence which has a high degree of similarity with the above sequence. For the purpose of the invention all RI active proteins having an amino acid sequence of at least 80% overlap with the above sequence are also embraced in the scope of the invention. More preferred is an overlap of at least 90%, most preferred more than 95%, e.g. those amino acid sequences which differ none or only one or two amino acids with the above sequence.
For the purpose of the invention the degree of overlap of two (partial) amino acid sequences can be calculated as follows:
(a) the two amino acid sequences are aligned and the number of amino acids which are identical and appear in the same order are counted (X)
(b) every change, deletion or addition of an amino acid is counted as 1 point, and the total of changes, deletions and additions is calculated (Y)
(c) the degree of overlap can now been calculated as X*100%/(X+Y).
For example the (partial) amino acid sequence from the N-terminus of: A-P-A-V-V-M-G-D-A-E-S-F-G-A-I-A-H-G-G-L (SEQ ID NO:2), can be aligned with the control as follows:
A-P-A-V-V-M-G-D-A-E-S-F-G-A-I-A-H-G-G-L (SEQ ID NO:2)
A-P-A-W -M- D-A-E-S-F-G-A-I-A-H-G-G-L (SEQ ID NO:1).
This leads to a total number of identical amino acids in the same order of 17. The number of changes is 1 (W into V at the fourth position); the number of additions is 2 (V at fifth position, G at 7th position), while there are no deletions. The total of changes, additions and deletions is therefore 3. This leads to a degree of overlap of 17*100%/(17+3)=85%
The protein having (partial) amino acid sequence from the N-terminus of:
A-P-A-V-V-M-G-D-A-E-S-F-G-A-I-A-H-G-G-L (SEQ ID NO:2) is hence also embraced within the invention.
Also embraced within the scope of the present invention are modified versions of the above described proteins whereby said modification does not materially affect the ice recrystallisation inhibition properties, such as glycosylated versions thereof.
For the purpose of the invention the term about 24 kDa molecular weight means any molecular weight from 20 to 28 kDa as measured on SDS-PAGE using standard reference markers, more preferably the molecular weight is from 22 to 26 kDa.
The advantageous AFP of the present invention can be derived from Lichen especially from the species Umbilicaria antarctica. 
Also embraced within the scope of the present invention are anti-freeze proteins which although originally derived from Lichen are produced by other methods, for example by genetic modification techniques whereby for example microorganisms or plants are genetically modified to produce the above described proteins. These proteins are also embraced within the term xe2x80x9ccan be derived from Lichenxe2x80x9d.
Also embraced within the scope of the present are nucleic acid sequences which are capable to encode the above described AFPs.
Vectors containing a nucleic acid sequence capable of encoding the AFP of the invention are also embraced within the scope of the invention.
Based on the above information it is also possible to genetically modify other natural sources such that they produce the advantageous AFP as identified here-above.
Applicants also have found that AFPs of the above sequence have improved ice-recrystallisation inhibition properties. A suitable test for determining the ice recrystallisation inhibition properties is described in the examples and involves the quick freezing to at least xe2x88x9240xc2x0 C., for example xe2x88x9280xc2x0 C. followed by storage for one hour at xe2x88x9260xc2x0 C. Preferably AFPs in accordance to the invention provide a ice particle size following an ice recrystallisation inhibition assayxe2x80x94as described in the examplesxe2x80x94of 15 VM or less, more preferred from 5 to 15 xcexcm.
The AFP of the invention can conveniently be used in food products, preferably in food products which are frozen or intended to be frozen. Especially preferred is the use of AFPs in products which are heated e.g. by pasteurisation or sterilisation prior to freezing. Especially preferred is the use in frozen confectionery products.
Examples of such food products are: frozen confectionery mixes such as ice-cream mixes and water-ice mixes which are intended to be pasteurised prior to freezing. Such mixes are usually stored at ambient temperature. Suitable product forms are for example: a powder mix which is packed for example in a bag or in sachets. Said mix being capable of forming the basis of the frozen food product e.g. after addition of water and optionally other ingredients andxe2x80x94optionalxe2x80x94aeration.
Another example of a suitable mix could be a liquid mix (optionally aerated) which, if necessary after addition of further components and optional further aeration can be frozen.
The clear advantage of the above mentioned mixes is that the presence of the AFP ingredient makes that the mixes can be frozen under quiescent conditions, for example in a shop or home freezer without the formation of unacceptable ice crystal shapes and hence with a texture different to products normally obtained via quiescent freezing.
Very conveniently these mixes are packed in closed containers (e.g. cartons, bags, boxes, plastic containers etc.). For single portions the pack size will generally be from 10 to 1000 g. For multiple portions pack sizes of up to 500 kg may be suitable. Generally the pack size will be from 10 g to 5000 g.
As indicated above the preferred products wherein the AFPs are used are frozen confectionery product such as ice-cream or water-ice. Preferably the level of AFPs is from 0.00001 to 0.5 wt % based on the final product. If dry-mixes or concentrates are used, the concentration may be higher in order to ensure that the level in the final frozen product is within the above ranges.
For the purpose of the invention the term frozen confectionery product includes milk containing frozen confections such as ice-cream, frozen yoghurt, sherbet, sorbet, ice milk and frozen custard, water-ices, granitas and frozen fruit purees. For some applications the use in fermented food products is less preferred.
Preferably a the level of solids in the frozen confection (e.g. sugar, fat, flavouring etc.) is more than 4 wt %, for example more than 30 wt %, more preferred from 40 to 70 wt %.
Frozen confectionery products according to the invention can be produced by any method suitable for the production of frozen confectionery. Especially preferably however all the ingredients of the formulation are fully mixed before pasteurisation and before the freezing process starts. The freezing process may advantageously involve a hardening step, for example to a temperature of xe2x88x9230 Fahrenheit or lower.